Ribonucleotide reductase inhibitors and methods of use

ABSTRACT

Provided herein are novel compounds that inhibit ribonucleotide reductase (RR) by binding to RRM2 and interfering with the activity of the RRM1/RRM2 holoenzyme, as well as methods of synthesizing these novel compounds. The compounds may be used to inhibit RR activity and to treat various conditions associated with RRM2 expression, such as for example certain cancer types, mitochondrial diseases, or degenerative diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/418,366, filed Jan. 27, 2017, which is a divisional of U.S.application Ser. No. 14/754,100, filed Jun. 29, 2015, now U.S. Pat. No.9,598,385, which is a continuation of U.S. application Ser. No.14/444,172, filed Jul. 28, 2014, now U.S. Pat. No. 9,126,960, which is acontinuation of International Application No. PCT/US2013/024490, filedFeb. 1, 2013, which claims the benefit of U.S. application Ser. No.13/364,263, filed Feb. 1, 2012, now U.S. Pat. No. 8,372,983, thedisclosure of each of which is incorporated by reference herein in itsentirety for all purposes.

GOVERNMENT INTEREST

This invention was made with Government support under grant numberCA127541 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND

Ribonucleotide diphosphate reductase (RR) is a highly regulated enzymein the deoxyribonucleotide synthesis pathway that is ubiquitouslypresent in human, bacteria, yeast, and other organisms (Jordan 1998). RRis responsible for the de novo conversion of ribonucleotide diphosphateto 2′-deoxyribonucleotide diphosphate, a process that is essential forDNA synthesis and repair (Thelander 1986; Jordan 1998; Liu 2006). RR isdirectly involved in tumor growth, metastasis, and drug resistance (Yen1994; Zhou 1995; Nocentini 1996; Fan 1998; Zhou 1998).

The proliferation of metastatic cancer cells requires excess dNTPs forDNA synthesis. Therefore, an increase in RR activity is necessary as ithelps provide extra dNTPs for DNA replication in primary and metastaticcancer cells. Because of this critical role in DNA synthesis, RRrepresents an important target for cancer therapy. However, there hasbeen little progress in the development of RR inhibitors for use incancer treatment. The three RR inhibitors currently in clinical use(hydroxyurea, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP),and GTI2040) each have significant drawbacks. Therefore, there is a needin the art for more effective compositions and methods for targeting andtreating RR-based cancers.

SUMMARY

In certain embodiments, a novel set of compounds including COH4, COH20,and COH29, as well as various chemical derivatives thereof, areprovided. Also provided are compositions and pharmaceutical formulationscomprising these compounds.

In certain embodiments, methods are provided for inhibiting RR activityin a cell by contacting the cell with one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

In certain embodiments, methods are provided for inhibiting the growthor proliferation of a cell expressing RRM2 by contacting the cell with atherapeutically effective amount of one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

In certain embodiments, methods are provided for treating cancer in asubject in need thereof by administering a therapeutically effectiveamount of one or more of the compounds provided herein, including COH4,COH20, and/or COH29. In certain embodiments, the cancer may becharacterized by RRM2 overexpression, and in certain embodiments thecancer may be resistant to treatment with hydroxyurea.

In certain embodiments, methods are provided for inhibitingproliferation of a stem cell expressing RRM2 by contacting the stem cellwith a therapeutically effective amount of one or more of the compoundsprovided herein, including COH4, COH20, and/or COH29.

In certain embodiments, a compound is provided having the formula:

In Structure VIA, R is substituted or unsubstituted aryl. R₁, R₂, R₃,R₄, R₅, R₆, and R₇ are independently hydrogen, —OH, —NH₂, —SH, —CN,—CF₃, —NO₂, oxo, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R₈,R₉, R₁₀ and R₁₁ are independently a hydroxyl protecting group. R₈ and R₉are optionally joined together to form a substituted or unsubstitutedheterocycloalkyl. R₁₀ and R₁₁ are optionally joined together to form asubstituted or unsubstituted heterocycloalkyl.

In certain embodiments, a composition is provided including the compoundof Structure VIA or embodiments thereof and an organic solvent (e.g. anon-polar solvent of a polar aprotic solvent).

In certain embodiments, a composition is provided including the compoundof Structure VIA or embodiments thereof and a hydroxyl deprotectingagent.

In certain embodiments, a method is provided for synthesizing a compoundhaving the structure:

The method includes contacting a hydroxyl deprotecting agent with acompound having the formula:

The R₈, R₉, R₁₀ and R₁₁ deprotecting groups are thereby removed. InStructure VIA, R is substituted or unsubstituted aryl. R₁, R₂, R₃, R₄,R₅, R₆, and R₇ are independently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃,—NO₂, oxo, halogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R₈, R₉,R₁₀ and R₁₁ are independently a hydroxyl protecting group. R₈ and R₉ areoptionally joined together to form a substituted or unsubstitutedheterocycloalkyl. R₁₀ and R₁₁ are optionally joined together to form asubstituted or unsubstituted heterocycloalkyl.

In certain embodiments, methods are provided for the synthesis and/orpurification of various compounds disclosed herein, including COH4,COH20, and/or COH29. In certain embodiments, methods are provided forsynthesizing and/or purifying COH29 or various COH29 synthesisintermediates. In certain of these embodiments, the synthesis methodsprovided herein utilize veratrole as a starting material, and in certainof these embodiments COH29 synthesis is accomplished via1-(3,4-dimethoxyphenyl)-2-phenylethanone,4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-amine, andN-(4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-yl)-3,4-dimethoxybenzamideintermediates. In certain of these embodiments, synthesis of COH29proceeds via the following synthesis pathway:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: Suppression of RRM2 via siRNA in mice implanted with humanHepG2 liver cancer cells decreases tumor growth. FIG. 1A: HP TVinjection of female BALB/c Mice with pR2Luc. FIG. 1B: Images taken2-days post-injection, all at same scale.

FIGS. 2A-2B: RRM2 overexpression in cancer tissues and cell lines incomparison to normal tissue. FIG. 2A: Histogram of M2 mRNA expressionlevel in normal (N) and breast cancer tissue (Y). FIG. 2B: Figuredepicts Western blot which revealed that normal human tissues other thanfetal liver and testis express low levels of RRM2, whereas cancer cellsexpress significantly higher levels of RRM2.

FIGS. 3A-3C: RRM2 transfectant enhances invasive potential in humancancer cell lines. Human oropharyngeal cancer KB (wild-type p53) andhuman prostate cancer PC3 (truncated p53) cells were transfected withsense RRM2 (KBM2 and PC3M2, respectively) and control vector, and theresulting overexpression of RRM2 was confirmed by Western blot analysis(FIG. 3A). Transfectants were applied to the upper layer of MATRIGEL® ina Borden chamber. After 72 hours, cells that invaded to the lower layerwere fixed with alcohol, stained with methylene blue, and counted andexamined (FIG. 3B). FIG. 3C: Comparison of RR expression and migrationability of Vector and RRM2 sense transfectants.

FIG. 4: Prediction of V-shaped ligand binding pocket on RRM2. Ironclusters are shown in red.

FIG. 5: Synthesis strategy for NCI-3 analogs

FIG. 6: Inhibition of RR activity in vitro by HU, 3-AP, NCI-3, COH4, andCOH20.

FIG. 7: Inhibition of intracellular RR activity by COH20.

FIG. 8: Inhibition of dNTP pools by COH20 in KB cells.

FIG. 9: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human prostateLNCaP cancer cells in vitro.

FIG. 10: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human KB cancercells in vitro.

FIG. 11: Cytotoxicity of 3-AP, HU, COH4, and COH20 in normal humanfibroblast (NHDF) cells in vitro.

FIG. 12: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human KBHUR cancercells in vitro.

FIG. 13: Cytotoxicity of 3-AP, HU, COH4, and COH20 in human KBMDR(multidrug resistant) cells in vitro.

FIG. 14: Inhibition of human KB and KBMDR cell proliferation by COH20.

FIGS. 15A-15H: Flow cytometry of KB cells following treatment with 3-APor COH20 at indicated concentrations. FIGS. 15E-15H: Annexin staining ofKB cells following treatment with 3-AP or COH20 at indicatedconcentrations.

FIG. 16: Single-dose pharmacokinetics of COH20 in rats.

FIG. 17: COH20 maximal tolerated dose determination in normal mice.

FIG. 18: Biacore analysis of RRM2 binding to COH20 and 3-AP.

FIG. 19: Interference of RRM1 binding to RRM2 by COH20.

FIG. 20: Inhibition of cancer cell growth by single-dose administrationof COH29.

FIG. 21: Inhibition of cancer cell growth by multiple-doseadministration of COH29.

FIGS. 22A-22I: Inhibition of cancer cell growth by multiple-doseadministration of COH29. Results are grouped by cell line tissue oforigin. Cell line legend for FIGS. 22A-22I: leukemia, non-small celllung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renalcancer, prostate cancer, breast cancer, respectively.

FIG. 23: Inhibition of cancer cell growth by multiple-doseadministration of COH29.

FIG. 24: Inhibition of cancer cell growth by multiple doseadministration of COH29.

FIG. 25: Pharmacokinetic of COH29 in rats.

FIG. 26: Synthesis of COH29.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

The following abbreviations are used herein: 3-AP,3-aminopyridine-2-carboxaldehyde thiosemicarbazone; DCM,dichloromethane; DMF, dimethylformamide; dNTP; deoxyribonucleotidetriphosphate; HU, hydroxyurea; RR, ribonucleotide reductase; RRM1,ribonucleotide reductase large subunit; RRM2, ribonucleotide reductasesmall subunit.

The phrase “therapeutically effective amount” as used herein refers toan amount of a compound that produces a desired therapeutic effect. Theprecise therapeutically effective amount is an amount of the compositionthat will yield the most effective results in terms of efficacy in agiven subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20^(th) edition, Williams & Wilkins PA, USA) (2000).

“Treating” or “treatment” of a condition as used herein may refer topreventing or alleviating a condition, slowing the onset or rate ofdevelopment of a condition, reducing the risk of developing a condition,preventing or delaying the development of symptoms associated with acondition, reducing or ending symptoms associated with a condition,generating a complete or partial regression of a condition, curing acondition, or some combination thereof. With regard to cancer,“treating” or “treatment” may refer to inhibiting or slowing neoplasticand/or malignant cell growth, proliferation, and/or metastasis,preventing or delaying the development of neoplastic and/or malignantcell growth, proliferation, and/or metastasis, or some combinationthereof. With regard to a tumor, “treating” or “treatment” may refer toeradicating all or part of a tumor, inhibiting or slowing tumor growthand metastasis, preventing or delaying the development of a tumor, orsome combination thereof.

A cancer “characterized by overexpression of RRM2” as used herein refersto any cancer type that expresses RRM2 at either the mRNA or proteinlevel at a level greater than that of a corresponding normal cell ortissue. For example, a prostate cancer cell line is a cancercharacterized by overexpression of RRM2 if it expresses RRM2 at eitherthe mRNA or protein level at a level greater than that observed in acorresponding normal prostate cell. A cancer characterized byoverexpression of RRM2 as used herein also refers to any cancer type inwhich RRM2 inhibitors exhibit additional or selective effects comparedto normal, untransformed cells or tissues. For example, a cancer type isa cancer characterized by RRM2 overexpression if it has a greaterdependency on the nucleotide pool because of a difference in mitoticindices with normal cells, making it more sensitive to RRM2 inhibition.

As used herein, the term “hydroxyl protecting group” is a monovalentchemical moiety covalently bound to a monovalent hydroxyl oxygen atomthat functions to prevent the hydroxyl moiety from reacting withreagents used in the chemical synthetic methods described herein(commonly referred to as “protecting” the hydroxyl group) and may beremoved under conditions that do not degrade the molecule of which thehydroxyl moiety forms a part (commonly referred to as “deprotecting” thehydroxyl group) thereby yielding a free hydroxyl. A hydroxyl protectinggroup can be acid labile, base labile, or labile in the presence ofother reagents. Hydroxyl protecting groups include but are not limitedto activated ethylene protecting group, a benzyl ether protecting group,a silicon-based carbonate protecting group, an acetal protecting groupor a cyclic acetal protecting group. Hydroxyl protecting groups includemethyl benzyl, p-methoxybenzyl, allyl, trityl, p-methoxyphenyl,tetrahydropyranyl, methoxymethyl, 1-ethoxyethyl, 2-methoxy-2-propy,2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl,2-trimethylsilylethoxymethyl, methylthiomethyl, trimethylsilyl,triethylsilyl, triisopropylsilyl, triphenylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl,

wherein n is 0 or 1; R₁₂ is hydrogen, —OH, —OR₁₆ or substituted orunsubstituted alkyl, wherein Rib is substituted or unsubstituted alkyl;and R₁₃, R₁₄ and R₁₅ are substituted or unsubstituted alkyl.

As used herein, the term “hydroxyl deprotecting agent” is a chemicalcompound or element that functions to remove a hydroxyl protectinggroup, thereby yielding a free hydroxyl. Hydroxyl deprotecting agentsuseful in the present methods include: zinc bromide, magnesium bromide,titanium tetrachloride, dimethylboron bromide, trimethylsilyl iodide,silver (Ag+) salts, mercury (Hg+) salts, zinc, samarium diiodide, sodiumamalgam, trifluoroacetic acid, hydrofluoric acid, hydrochloric acid,hydrogenation, (TBAF) tetra-n-butylammonium fluoride, boron trifluoride,silicon tetrafluoride, boron tribromide, an aryl methyl ether,tetrabutylammonium fluoride, hydrogen/Pd/C, Zn/acid or ammonia.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. An alkoxy is an alkyl attached to the remainder of the moleculevia an oxygen linker (—O—).

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom selected from the group consisting of O, N, P, Si, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized,and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) 0, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. Non-limiting examples of heteroaryl groups includepyridinyl, pyrimidinyl, thiophenyl, furanyl, indolyl, benzoxadiazolyl,benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl,indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl,benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothiophenyl,phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl,oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl,benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl,oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl,benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl,pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. Theexamples above may be substituted or unsubstituted and divalent radicalsof each heteroaryl example above are non-limiting examples ofheteroarylene.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms (asindicated herein) of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SW, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike). Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″,R′″, and R″″ are preferably independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupswhen more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from: oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and/or each substituted or unsubstituted heterocycloalkyl isa substituted or unsubstituted 3 to 8 membered heterocycloalkyl.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and/or each substitutedor unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to7 membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

The symbol

represents the point of attachment of a substituent to the remainder ofthe compound.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

Description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

dNTP production in eukaryotes is tightly regulated by RR, whichcatalyzes the rate-limiting step in deoxyribonucleotide synthesis(Jordan 1998). RR consists of a large subunit and a small subunit. Inhumans, one large subunit (RRM1, also referred to as M1) and two smallsubunits (RRM2, also referred to as M2, and p53R2) have been identified(Tanaka 2000; Liu 2006). The small RR subunits form two equivalentdinuclear iron centers that stabilize the tyrosyl free radical requiredfor initiation of electron transformation during catalysis (Ochiai 1990;Cooperman 2003; Liu 2006).

RRM1 is a 170 Kd dimer containing substrate and allosteric effectorsites that control RR holoenzyme activity and substrate specificity(Cory 1983; Wright 1990; Cooperman 2003; Liu 2006).

RRM2 is an 88 Kd dimer containing a tyrosine free radical and a non-hemeiron for enzyme activity (Chang 1979). p53R2 contains a p53-binding sitein intron 1 and encodes a 351-amino-acid peptide with strikingsimilarity to RRM2 (Tanaka 2000). RRM2 and p53R2 have an 80% similarityin amino acid sequence (Tanaka 2000).

p53R2 has been identified as a transcriptional target of p53 (Nakano2000; Tanaka 2000; Yamaguchi 2001), while RRM2 is transcriptionallyregulated by cell cycle-associated factors such as NF-Y and E2F (Filatov1995; Currie 1998; Chabes 2004; Liu 2004). Therefore, expression ofp53R2, but not RRM2, is induced by ultraviolet (UV) light,gamma-irradiation, or Doxorubicin (Dox) treatment in a p53-dependentmanner (Lozano 2000; Tanaka 2000; Guittet 2001). In p53 mutant ordeleted cells, RRM2 can replace p53R2 in the DNA repair process forcells exposed to UV irradiation (Zhou 2003; Liu 2005). P53R2 has beenfound to have a stronger reducing capacity than RRM2, which may providea chaperone effect in stabilizing p21 (Xue 2007).

It has been observed that the RB tumor suppressor suppresses RR subunitsas a mechanism for regulating cell cycle progression (Elledge 1990). RBinactivation, often observed in tumors, leads to increased dNTP levelsand a concomitant resistance of tumor cells to drugs such as5-fluorouracil (5-Fu) and HU (Angus 2002). While overexpression of theRRM2 subunit promotes transformation and tumorigenic potential via itscooperation with several activated oncogenes (Fan 1998), overexpressionof the RRM1 subunit suppresses malignant potential in vivo (Fan 1997).Increased expression of RRM2 has been found to increase thedrug-resistant properties of cancer cells and to increase invasivepotential, whereas RRM2 suppression leads to the reversal of drugresistance and results in decrease proliferation of tumor cells (Zhou1995; Huang 1997; Zhou 1998; Goan 1999; Chen 2000; Nakano 2000; Kuschak2002). Normal cells express very low levels of RR in non-proliferativestatus, whereas most neoplastic cells overexpress RR, thereby supplyingdNTP pools for DNA synthesis and cell proliferation. Thus, specificinhibition of RRM2 is likely to provide antineoplastic benefits.

Although RR represents an important target for cancer therapy, there areonly three RR inhibitors in clinical use: hydroxyurea (HU),3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, TRIAPINE®),and GTI2040. HU, which blocks DNA synthesis by reducing the tyrosyl freeradical, has been marketed as a cancer therapeutic for over 30 years andis the only RR inhibitor that is commercially available (Nocentini1996). However, resistance to HU treatment is a common problem (Lassmann1992; Nyholm 1993; Le 2002). 3-AP, a small molecule iron chelatorinactivates RR, has been found to cause hypoxia, respiratory distress,and methemoglobulin of red blood cells. In addition, 3-AP selectivelytargets p53R2 instead of RRM2. GTI2040, an antisense molecule, has thusfar been ineffective in human trials. Other issues relating to thesethree RR inhibitors are incomplete RR blocking, short half-life, andregeneration of RR. In addition, mutation of p53R2 results in hereditarymitochondria depletion syndrome, but not cancer, and p53R2 knockout micedemonstrate kidney tubule disorder but no obvious cancer growth (Kimura2003). These observations suggest that RRM2 is responsible for tumorproliferation and metastatic potential, whereas p53R2 induced by DNAdamage signals for DNA repair. Therefore, an ideal RR inhibitor for usein cancer therapy would have greater potency than HU, less ironchelating ability than 3-AP, and specific targeting of RRM2.

As disclosed herein, RRM2 has been validated as an anti-cancer target.Suppression of RRM2 via siRNA was found to decrease tumor growth in miceimplanted with human HepG2 liver cancer cells. Expression of RRM2 wasdetermined to be significantly higher in cancer cells than incorresponding normal cells. In addition, human KB and PC3 cellstransfected with RRM2 exhibited increased invasive potential versustheir non-transfected counterparts, suggesting that RRM2 enhances theinvasive potential of cancer cells.

A diverse compound library from NCI Developmental Therapeutics Program(DTP) was screened to identify compounds that inhibit RR. Three of thefour compounds identified in this screen that inhibited RRM1/RRM2activity by 80% of more shared a similar structural scaffold, NCI-3.NCI-3 has the structure:

Initial hits from the screening process were synthetically andrationally optimized to obtain the RR inhibitors COH1, COH2, COH4,COH20, and COH29, the structures of which are set forth below.

Each of these compounds exhibited the ability to inhibit RR to asignificant degree, and COH20 and COH29 both exhibited the ability toinhibit cancer cell growth across a wide range of cancer cell types.Accordingly, in certain embodiments the present application disclosesnovel RR inhibitors, compositions, formulations, and kits comprising oneor more of these inhibitors, and methods of using these inhibitors toinhibit RR, inhibit cell growth or proliferation, treat cancer, and/orinhibit stem cell proliferation.

COH20 consists of three basic structural units: a pharmacophore (forinhibition of RR activity), a binding group (for selectivity), and alinking group (to connect the pharmacophore and the binding group. Asdisclosed herein, COH20 exhibited low micromolar range inhibition ofboth recombinant and intracellular RR in vitro, and caused a decrease indNTP pools. Biochemical analysis revealed that COH20 targets RRM2. Uponbinding the RRM1/RRM2 complex, COH20 appears to reside at the V-shapedpocket at the interface between RRM1 and RRM2 and block the free radicaltransfer pathway though a novel catechol radical stabilizationmechanism. Considering the size and chemical composition of COH20 andthe distance to the dinuclear iron center, the bound ligand (in thispocket) does not appear to be susceptible to iron chelation as with 3-APor involved in the direct quenching of the initially formed tyrosyl freeradical as with HU. COH20 was found to inhibit growth of the humanleukemia cell lines REH and MOLT-4, the human prostate cancer cell lineLNCaP, and the human oropharyngeal cancer cell line KB in vitro at aconcentration of less than 10 μM, while exhibiting less cytotoxicitytowards normal fibroblast cells than HU. COH20 also exhibited greatercytotoxicity towards the HU-resistant cell line KBHURs than 3-AP,indicating that it is capable of overcoming HU drug resistance. COH20exhibited cytotoxicity towards KBMDR cells at lower concentration than3-AP or HU (80 μM versus 200 μM and >1000 μM, respectively), indicatingthat COH20 circumvents MDR more effectively than 3-AP. In addition,COH20 had very low toxicity when administered to mice, with no evidenceor iron chelation or methemoglobulin.

As disclosed herein, COH29 showed promising growth inhibitory effects ona wide range of human cancer cell lines, including:

-   -   human non-small cell lung cancer cell lines NCI-H23, NCI-H522,        A549-ATCC, EKVX, NCI-H226, NCI-H332M, H460, H0P62, HOP92;    -   human colon cancer cell lines HT29, HCC-2998, HCT116, SW620,        COLO205, HCT15, KM12;    -   breast cancer cell lines MCF7, MCF7ADRr, MDAMB231, HS578T,        MDAMB435, MDN, BT549, T47D;    -   ovarian cancer cell lines OVCAR3, OVCAR4, OVCAR5, OVCAR8,        IGROV1, SKOV3;    -   human leukemia cell lines CCRFCEM, K562, MOLT4, HL60, RPMI8266,        SR;    -   renal cancer cell lines UO31, SN12C, A498, CAKI1, RXF393, 7860,        ACHN, TK10;    -   melanoma cell lines LOXIMVI, MALME3M, SKMEL2, SKMEL5, SKMEL28,        M14, UACC62, UACC257;    -   prostate cancer cell lines PC3, DU145; and    -   CNS cancer cell lines SNB19, SNB75, U251, SF268, SF295, SM539.

In certain embodiments, COH29 showed a GI50 of less than about 10 μM forthe NCI 60 human cancer cell lines, except colon cancer cell line HT29,melanoma cancer cell line UACC-257, ovarian cancer cell lineNCI/ADR-RES, and renal cancer cell line CAKI-1. In addition,pharmacokinetic studies of COH29 showed a dose-dependent manner whenCOH29 was administered by i.v. bolus.

COH4, COH20 and COH29 represent unique RR inhibitors with highantitumoral activity that provide significant advantages over previouslydisclosed RR inhibitors. Specifically, COH20 offers a unique mechanismand target specificity that interferes with the radical transfer pathwayat the RRM1/RRM2 interface with greater potency than HU and ameliorationof the iron chelation-related side effects observed with 3-AP.Therefore, provided herein in certain embodiments are small molecule RRinhibitors and methods of using these inhibitors to inhibit RR and totreat cancer. The inhibitors disclosed herein are capable of overcomingHU resistance, a common obstacle to cancer therapy, and are also capableof overcoming multidrug resistance.

In certain embodiments, a novel RR inhibitor as disclosed herein isCOH1, COH2, COH4, COH20, or COH29, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or prodrug derivative thereof.

In certain embodiments, a novel RR inhibitor as disclosed herein is:

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrugderivative thereof, wherein:

R₁ and R₂ are independently selected from the group consisting ofhydrogen, alkyl, and aryl groups;

R₃ is selected from the group consisting of alkyl groups; and

X is selected from the group consisting of Br, halogen, alkyl and arylgroups.

In certain embodiments, an RR inhibitor as disclosed herein is:

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrugderivative thereof, wherein

X is selected from the group consisting of halogen, substituted andunsubstituted alkyl and substituted and unsubstituted aryl groups;

R₁-R₂ are independently selected from the group consisting of H, OH,substituted and unsubstituted alkyl, and substituted and unsubstitutedaryl groups; and

R₁-R₂ may combine together to form a ring wherein the ring is aryl ornon-aryl.

As used herein, the term “halogen” means F, Cl, Br, I and At.

The RR inhibitors disclosed herein specifically target RRM2, inhibitingthe interaction between RRM1 and RRM2 and inhibiting the activity of theRR complex. Therefore, these inhibitors may be used to certain cancers,including cancers associated with the overexpression of RRM2 or forwhich there exists a suitable therapeutic index. As disclosed herein,RRM2 may be overexpressed in breast cancer cells versus normal cells,and expression of exogenous RRM2 increases cancer cell invasivepotential. The inhibitors disclosed herein have been shown to inhibitgrowth of multiple cancer cell types in vitro, supporting the use ofthese inhibitors to treat a wide range of cancers.

Previous studies have shown that RRM2 is highly expressed in stem cellsin the colon (Liu 2006). At the early stages of colon cancer, RRM2expression decreases slightly. However, RRM2 expression increasessignificantly once the tumor becomes aggressive. These results supportthe findings herein that RRM2 expression is associated with an increasein cancer cell invasiveness. In addition, they support the use of theinhibitors disclosed herein to inhibit the growth or proliferation ofstem cells giving rise to cancer, thereby preventing or slowing theonset of certain cancer types.

In addition to cancer, the inhibitors disclosed herein may be used totreat other conditions associated with RR or RR overexpression, such asfor example various mitochondrial, redox-related, or degenerativediseases. In addition, the inhibitors may be used to inhibit growth orproliferation of cells expressing RR.

Provided herein in certain embodiments are methods for both small- andlarge-scale synthesis of the small molecule RR inhibitors disclosedherein. In certain of these embodiments, the small molecule RR inhibitorbeing synthesized is COH29. Small molecule RR inhibitors synthesizedusing the methods provided herein may be purified by various methodsknown in the art. Specific purification steps that may be utilizedinclude, for example, precipitation, trituration, crystallization, orchromatographic techniques such as silica gel chromatography.

In certain embodiments, methods are provided for the synthesis of COH29.In certain of these embodiments, the starting material is a compoundhaving the structure:

where R₁, R₂, and R₃ are each independently hydrogen, a substituted orunsubstituted aryl, or a substituted or unsubstituted alkyl. In certainof these embodiments, the starting material compound is veratrole(1,2-dimethoxybenzene), which has the structure:

In certain embodiments, the first step in the synthesis of COH29 is theconversion of the starting material compound to a first intermediatecompound having the structure:

where R is a substituted or unsubstituted aryl, including for example aphenyl, and R₁, R₂, and R₃ are each independently hydrogen, asubstituted or unsubstituted aryl, or a substituted or unsubstitutedalkyl. In certain of these embodiments, the first intermediate compoundis 1-(3,4-dimethoxyphenyl)-2-phenylethanone, which has the structure:

In certain embodiments, the starting material compound is converted tothe first intermediate compound by adding the starting material compoundto a mixture comprising anhydrous AlCl₃ in dichloromethane (CH₂Cl₂) anda compound having the structure:

where R is a substituted or unsubstituted aryl, including for example aphenyl. In certain embodiments, the compound of Structure IV isphenylacetyl chloride. In certain embodiments, the first intermediatecompound is precipitated from the combined organic layers, for exampleusing dichloromethane and/or hexanes. In certain embodiments, the firststep of a COH29 synthesis method as provided herein is summarized asfollows:

In certain embodiments, the second step in the synthesis of COH29 is theconversion of a first intermediate compound of Structure III to a secondintermediate compound having the structure:

where R is a substituted or unsubstituted aryl, including for example aphenyl, and R₁, R₂, R₃, and R₇ are each independently hydrogen, asubstituted or unsubstituted aryl, or a substituted or unsubstitutedalkyl. In certain of these embodiments, the second intermediate compoundis 4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-amine which has thestructure:

In certain embodiments, the first intermediate compound is converted tothe second intermediate compound by dissolving the first intermediatecompound and pyridinium tribromide in dichloromethane, followed bywashing and drying. In certain embodiments, the dried reaction mixtureis then mixed with thiourea in ethanol. In certain embodiments, theresultant product can be concentrated, washed, and purified bytrituration and drying.

In certain embodiments, the second step of a COH29 synthesis method asprovided herein is summarized as follows:

In certain embodiments, the third step in the synthesis of COH29 is theconversion of a second intermediate compound of Structure V to a thirdintermediate compound having the structure:

where R is a substituted or unsubstituted aryl, including for example aphenyl, and R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are each independentlyhydrogen, a substituted or unsubstituted aryl, or a substituted orunsubstituted alkyl. In certain of these embodiments, the secondintermediate compound isN-(4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-yl)-3,4-dimethoxybenzamide,which has the structure:

In certain embodiments, a second intermediate compound is converted to athird intermediate compound by adding the second intermediate compound,4-dimethylamino pyridine, and Et₃N to an acid chloride solutioncontaining 3,4-dimethoxybenzoic acid and dimethylformamide treated withthionyl chloride. In certain embodiments, the reaction may be quenchedwith NaHCO₃. In certain embodiments, the resultant suspension may beextracted with dichloromethane, and in certain of these embodiments theextracted product may be triturated using hexanes.

In certain embodiments, the third step of a COH29 synthesis method asprovided herein is summarized as follows:

In certain embodiments, the fourth step in the synthesis of COH29 is theconversion of a third intermediate compound of Structure VI to COH29. Incertain of these embodiments, the third intermediate compound is mixedwith boron tribromide in toluene, and in certain of these embodimentsthe reaction is quenched with ethanol, followed by precipitation fromwater. In certain embodiments, the resultant product undergoes one ormore purification steps. In certain of these embodiments, the product ispurified using a C-18 silica gel, optionally followed bycrystallization.

In certain embodiments, the fourth step of a COH29 synthesis method asprovided herein is summarized as follows:

In certain embodiments, synthesis of COH29 results in a final yield of50% or greater, and in certain of these embodiments the final yield is60% or greater, 70% or greater, 80% or greater, or 90% or greater.

In certain embodiments, methods are provided for the large-scalesynthesis of COH29 using the synthetic pathway set forth in FIG. 26.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

In certain embodiments, a compound is provided having the formula:

In Structure VIA, R is substituted or unsubstituted aryl. Inembodiments, R is unsubstituted phenyl. R₁, R₂, R₃, R₄, R₅, R₆, and R₇are independently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R₇ is not —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo or halogen.R₈, R₉, R₁₀ and R₁₁ are independently a hydroxyl protecting group. R₈and R₉ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl (e.g. 5 membered heterocycloalkyl). R₁₀and R₁₁ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl (e.g. 5 membered heterocycloalkyl). R₁,R₂, R₃, R₄, R₅, R₆, and R₇ may also independently be hydrogen,substituted or unsubstituted aryl (e.g. phenyl), or a substituted orunsubstituted alkyl (e.g. C₁-C₁₀ alkyl).

Where R₁, R₂, R₃, R₄, R₅, R₆, or R₇ are a substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl or substituted heteroaryl, the substituent may be asubstituent group, as defined above. The substituent may also be asize-limited substituent group, as defined above. The substituent mayalso be a lower substituent group, as defined above.

Where R is a substituted aryl, the substituent may be a substituentgroup, as defined above. The substituent may also be a size-limitedsubstituent group, as defined above. The substituent may also be a lowersubstituent group, as defined above.

In embodiments, R₈, R₉, R₁₀ and R₁₁ are independently substituted orunsubstituted alkyl (e.g. C₁-C₁₀ alkyl), substituted or unsubstitutedheteroalkyl (e.g. 2 to 10 membered heteroalkyl), substituted orunsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl), substituted orunsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),substituted or unsubstituted aryl (e.g. phenyl), or substituted orunsubstituted heteroaryl (e.g. 5 or 6 membered heteroaryl). Inembodiments, R₈, R₉, R₁₀ and R₁₁ are substituted or unsubstituted alkyl(e.g. C₁-C₁₀ alkyl), substituted or unsubstituted heteroalkyl (e.g. 2 to10 membered heteroalkyl), or substituted or unsubstituted aryl (e.g.phenyl). In embodiments, R₈, R₉, R₁₀ and R₁₁ are independently asubstituted silyl. A “substituted silyl” as used herein, refers to asubstituted heteroalkyl wherein at least one of the heteroatoms is asilicon atom. The silicon atom may be directly attached to the oxygenatom of the hydroxyl that is protected thereby forming a silyl ether. Inembodiments, the substituted silyl is substituted with an unsubstitutedalkyl (e.g. C₁-C₅ alkyl), unsubstituted heteroalkyl (e.g. 2 to 5membered heteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₆ cycloalkyl),unsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),unsubstituted aryl (e.g. phenyl), or unsubstituted heteroaryl (e.g. 5 or6 membered heteroaryl). In embodiments, the substituted silyl issubstituted with an unsubstituted alkyl (e.g. C₁-C₅ alkyl),unsubstituted aryl (e.g. phenyl), or unsubstituted heteroaryl (e.g. 5 or6 membered heteroaryl). In related embodiments, the silicon atom of thesubstituted silyl is directly attached to the oxygen atom of theremainder of the compound thereby forming a silyl ether.

Where R₈, R₉, R₁₀ and R₁₁ are a substituted alkyl, substitutedheteroalkyl (e.g. substituted silyl), substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl or substitutedheteroaryl, the substituent may be a substituent group, as definedabove. The substituent may also be a size-limited substituent group, asdefined above. The substituent may also be a lower substituent group, asdefined above.

R₁, R₂, R₃, R₄, R₅, R₆, and R₇ may also independently be hydrogen, —OH,—NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₁₇-substituted orunsubstituted alkyl (e.g. C₁-C₁₀ alkyl), R₁₇-substituted orunsubstituted heteroalkyl (e.g. 2 to 10 membered heteroalkyl),R₁₇-substituted or unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),R₁₇-substituted or unsubstituted heterocycloalkyl (e.g. 3 to 6 memberedheterocycloalkyl), R₁₇-substituted or unsubstituted aryl (e.g. phenyl),or R₁₇-substituted or unsubstituted heteroaryl (e.g. 5 or 6 memberedheteroaryl). In embodiments, R₇ is not —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,oxo or halogen. R₈ and R₉ may optionally be joined together to form aR₁₈-substituted or unsubstituted heterocycloalkyl (e.g. 5 memberedheterocycloalkyl). R₁₀ and R₁₁ may optionally be joined together to forma R₁₉-substituted or unsubstituted heterocycloalkyl (e.g. 5 memberedheterocycloalkyl). R may be R_(19A)-substituted or unsubstituted aryl.R₈, R₉, R₁₀ and R₁₁ may be R_(19B)-substituted or unsubstituted alkyl(e.g. C₁-C₁₀ alkyl), R_(19B)-substituted or unsubstituted heteroalkyl(e.g. 2 to 10 membered heteroalkyl), R_(19B)-substituted orunsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl), R_(19B)-substituted orunsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),R_(19B)-substituted or unsubstituted aryl (e.g. phenyl), orR_(19B)-substituted or unsubstituted heteroaryl (e.g. 5 or 6 memberedheteroaryl).

R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ may independently be R_(19C)-substituted orunsubstituted alkyl (e.g. C₁-C₁₀ alkyl), R_(19C)-substituted orunsubstituted heteroalkyl (e.g. 2 to 10 membered heteroalkyl),R_(19C)-substituted or unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),R_(19C)-substituted or unsubstituted heterocycloalkyl (e.g. 3 to 6membered heterocycloalkyl), R_(19C)-substituted or unsubstituted aryl(e.g. phenyl), or R_(19C)-substituted or unsubstituted heteroaryl (e.g.5 or 6 membered heteroaryl). R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ mayindependently be R_(19C)-substituted or unsubstituted alkyl (e.g. C₁-C₁₀alkyl), or R_(19C)-substituted or unsubstituted aryl (e.g. phenyl).

Each R₁₇, R₁₈, R₁₉, R_(19A), R_(19B) and R_(19C) are independently —OH,—NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₂₀-substituted orunsubstituted alkyl (e.g. C₁-C₁₀ alkyl), R₂₀-substituted orunsubstituted heteroalkyl (e.g. 2 to 10 membered heteroalkyl),R₂₀-substituted or unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),R₂₀-substituted or unsubstituted heterocycloalkyl (e.g. 3 to 6 memberedheterocycloalkyl), R₂₀-substituted or unsubstituted aryl (e.g. phenyl),or R₂₀-substituted or unsubstituted heteroaryl (e.g. 5 or 6 memberedheteroaryl). Each R₂₀ is independently —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,oxo, halogen, R₂₁-substituted or unsubstituted alkyl (e.g. C₁-C₁₀alkyl), R₂₁-substituted or unsubstituted heteroalkyl (e.g. 2 to 10membered heteroalkyl), R₂₁-substituted or unsubstituted cycloalkyl (e.g.C₃-C₈ cycloalkyl), R₂₁-substituted or unsubstituted heterocycloalkyl(e.g. 3 to 6 membered heterocycloalkyl), R₂₁-substituted orunsubstituted aryl (e.g. phenyl), or R₂₁-substituted or unsubstitutedheteroaryl (e.g. 5 or 6 membered heteroaryl). Each R₂₁ is independently—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl (e.g.C₁-C₁₀ alkyl), unsubstituted heteroalkyl (e.g. 2 to 10 memberedheteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),unsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),unsubstituted aryl (e.g. phenyl) or unsubstituted heteroaryl (e.g. 5 or6 membered heteroaryl).

In embodiments, each R₁₇, R₁₈, R₁₉, R_(19A), R_(19B) and R_(19C) areindependently —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen,unsubstituted alkyl (e.g. C₁-C₁₀ alkyl), unsubstituted heteroalkyl (e.g.2 to 10 membered heteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈cycloalkyl), unsubstituted heterocycloalkyl (e.g. 3 to 6 memberedheterocycloalkyl), unsubstituted aryl (e.g. phenyl), or unsubstitutedheteroaryl (e.g. 5 or 6 membered heteroaryl).

In embodiments, R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are independentlyhydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstitutedalkyl (e.g. C₁-C₁₀ alkyl), unsubstituted heteroalkyl (e.g. 2 to 10membered heteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),unsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),unsubstituted aryl (e.g. phenyl), or unsubstituted heteroaryl (e.g. 5 or6 membered heteroaryl). In embodiments, at least one of R₁, R₂, R₃, R₄,R₅, R₆, and R₇ is hydrogen. In embodiments, at least two of R₁, R₂, R₃,R₄, R₅, R₆, and R₇ are hydrogen. In embodiments, at least three of R₁,R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen. In embodiments, at least fourof R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen. In embodiments, at leastfive of R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen. In embodiments, atleast six of R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen. Inembodiments, R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen.

In embodiments, R₈, R₉, R₁₀ and R₁₁ are independently unsubstitutedalkyl (e.g. C₁-C₁₀ alkyl), unsubstituted heteroalkyl (e.g. 2 to 10membered heteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl),unsubstituted heterocycloalkyl (e.g. 3 to 6 membered heterocycloalkyl),unsubstituted aryl (e.g. phenyl), or unsubstituted heteroaryl (e.g. 5 or6 membered heteroaryl).

In embodiments, R₈, R₉, R₁₀ and R₁₁ are independently an activatedethylene protecting group, a benzyl ether protecting group, asilicon-based carbonate protecting group, or a cyclic acetal protectinggroup. In embodiments, R₈, R₉, R₁₀ and R₁₁ are methyl benzyl,p-methoxybenzyl, allyl, trityl, p-methoxyphenyl, tetrahydropyranyl,methoxymethyl, 1-ethoxyethyl, 2-methoxy-2-propy,2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl,2-trimethylsilylethoxymethyl, methylthiomethyl, trimethylsilyl,triethylsilyl, triisopropylsilyl, triphenylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl,

The symbol n is 0 or 1. R₁₂ is hydrogen, —OH, —OR₁₆, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In embodiments, R₁₂ is hydrogen, —OH, —OR₁₆ orsubstituted or unsubstituted alkyl. In embodiments, R₁₂ is hydrogen. R₁₆is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiments, R₁₆ issubstituted or unsubstituted alkyl. R₁₃, R₁₄ and R₁₅ are independentlysubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiment, R₁₃, R₁₄ and R₁₅are independently substituted or unsubstituted alkyl or substituted orunsubstituted aryl.

Where R₁₂, R₁₃, R₁₄ R₁₅ and R₁₆ is a substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl or substituted heteroaryl, the substituent may be asubstituent group, as defined above. The substituent may also be asize-limited substituent group, as defined above. The substituent mayalso be a lower substituent group, as defined above.

In embodiments, R₁₂ is hydrogen, methyl or —OCH₃. R₁₃, R₁₄ and R₁₅ maybe unsubstituted C₁-C₅ alkyl or unsubstituted phenyl. In embodiments,R₁₃, R₁₄ and R₁₅ may be unsubstituted C₁-C₅ alkyl.

In embodiments, R is unsubstituted aryl (e.g. phenyl). R₁, R₂, R₃, R₄,R₅, R₆, and R₇ may independently be hydrogen, unsubstituted aryl (e.g.phenyl), or unsubstituted alkyl (e.g. C₁-C₅ alkyl).

In embodiments, R₈ and R₉ are optionally joined together to form anacetal protecting group and R₁₀ and R₁₁ are optionally joined togetherto form an acetal protecting group. An “acetal protecting group” is usedaccording to its common meaning in the art of chemical synthesis, inwhich an acetal group is frequently used to protect catechol hydroxylmoieties. An acetal protecting group may have the formula:

In Structure VIC, R₂₂ and R₂₃ are independently hydrogen, substituted orunsubstituted alkyl (e.g. C₁ to C₅ alkyl, such as methyl) or substitutedor unsubstituted aryl (e.g. phenyl). The symbol

represents the point of attachment to the catechol oxygen atoms (i.e.oxygen atoms attached to R₈, R₉ and R₁₀ and R₁₁, respectively). Inembodiments, R₂₂ and R₂₃ are independently hydrogen, unsubstituted alkyl(e.g. C₁ to C₅ alkyl, such as methyl) or unsubstituted aryl (e.g.phenyl). In some embodiments, R₂₂ and R₂₃ are hydrogen. In embodiments,R₂₂ and R₂₃ are unsubstituted alkyl (e.g. C₁ to C₅ alkyl). Inembodiments, R₂₂ and R₂₃ are methyl. In some embodiments, R₂₂ and R₂₃are unsubstituted aryl (e.g. phenyl). In embodiments, the acetalprotecting group is a diphenyl methylene acetal.

In embodiments, the compound of Structure VIA has the formula:

In Structure VIB, R, R₈, R₉, R₁₀ and R₁₁ are as defined above, includingall embodiments thereof. In embodiments of the compound of Structure VIAor VIB, R is unsubstituted aryl. In embodiments of Structure VI or VIB,R is unsubstituted phenyl.

In embodiments, a composition is provided including the compound ofStructure VIA or embodiments thereof, or Structure VIB or embodimentsthereof, and an organic solvent (e.g. a non-polar solvent of a polaraprotic solvent). In embodiments, the organic solvent is a non-polarsolvent (e.g. toluene or 1,4-dioxane). In embodiments, the organicsolvent is dioxane. In embodiments, the organic solvent is a polaraprotic solvent (e.g. acetone, dimethylformamide, acetonitrile ofdimethyl sulfoxide). In embodiments, the organic solvent is dimethylformamide.

In embodiments, a composition is provided including the compound ofStructure VIA or embodiments thereof, or Structure VIB or embodimentsthereof and a hydroxyl deprotecting agent. The hydroxyl deprotectingagent is a chemical agent useful in removing a hydroxyl protecting groupas described herein. Useful chemical agents are selected as hydroxyldeprotecting agents to minimize degradation of the compound of StructureVIA and embodiments thereof, Structure VIB and embodiments thereof andStructure VID and embodiments thereof. In embodiments, the deprotectingagent is a reducing agent, an acidic agent or a basic agent. Inembodiments, the deprotecting agent is boron tribromide, an aryl methylether, tetrabutylammonium fluoride, a reducing agent (e.g. a palladiumreducing agent such as hydrogen/Pd/C), a metal acid agent (e.g. aZn/acid such as Zn/acetic acid or Zn/HCl) or ammonia.

In certain embodiments, a method is provided for synthesizing a compoundhaving the structure:

The method includes contacting a hydroxyl deprotecting agent (asdescribed above) with a compound having the formula:

In Structure VIA and Structure VID, R, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,R₉, R₁₀ and R₁₁ are as defined above, including all embodiments. Inembodiments, R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are independently hydrogen,substituted or unsubstituted aryl, or a substituted or unsubstitutedalkyl. R₈, R₉, R₁₀ and R₁₁ may independently be a hydroxyl protectinggroup. R₈ and R₉ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl. R₁₀ and R₁₁ are optionally joinedtogether to form a substituted or unsubstituted heterocycloalkyl.

EXAMPLES Example 1: Confirmation of RRM2 as a Target for Anti-CancerTherapy

An RRM2-luciferase fusion construct (“pR2Luc”) was administered alone orin combination with an RRM2 siRNA (“siR2B+5”) by hydrodynamic tail veininjection (HPTV) [plasmid 0.25 mg/kg, siRNA 1.25 mg/kg] to female BALB/cmice implanted with human HepG2 liver cancer cells. In vivobioluminescence imaging revealed potent down-regulation within mouseliver cancer cells over multiple weeks (FIGS. 1A-1B). Mice receivingRRM2 in combination with RRM2 siRNA exhibited a significant decrease intumor growth versus mice receiving RRM2 alone or in combination withcontrol siRNA (“siCONTROL”). These results suggest that RRM2 playscritical role in cancer growth and validates it as a therapeutic target.

RRM2 mRNA expression levels were measured by RT-PCR in 35 human freshfrozen breast cancer biopsies and corresponding normal tissue samples.Optimal PCR primer and probe concentrations of RRM2 and (3-actinhousekeeping gene were determined to reach maximum efficiency during theamplification. The PCR reaction was performed in a 20 μl final volume,adding 1 μL cDNA from each sample using TAQMAN® PCR mix (AppliedBiosystems, Foster City, Calif.). A significant increase in RRM2expression was observed in breast cancer tissue versus correspondingnormal tissue (p<0.05) (FIG. 2A). A Western blot revealed that normalhuman tissues other than fetal liver and testis express low levels ofRRM2, whereas cancer cells express significantly higher levels of RRM2(FIG. 2B).

Human oropharyngeal cancer KB (wild-type p53) and human prostate cancerPC3 (truncated p53) cells were transfected with sense RRM2 (KBM2 andPC3M2, respectively) and control vector, and the resultingoverexpression of RRM2 was confirmed by Western blot analysis.Transfectants were applied to the upper layer of MATRIGEL® in a Bordenchamber. After 72 hours, cells that invaded to the lower layer werefixed with alcohol, stained with methylene blue, and counted andexamined. RRM2 transfected cells exhibited increased invasive potentialin comparison to non-transfected cells (FIGS. 3A-3C).

Example 2: Identification of Novel RR Inhibitors

A diverse compound library from NCI Developmental Therapeutics Program(DTP) was subjected to a virtual screening process to identify potentialRR inhibitors. The DTP library contains 2,000 different compounds. Anovel ligand binding pocket on human RRM2 identified from the X-raycrystal structure (PDB 2UW2) was selected to identify potentialinhibitor compounds that were in close proximity to the RRM1/RRM2interface but distant from the dityrosyl-diiron center in order to avoidiron chelating side effects. This ligand binding pocket, which consistsof 32 amino acid residues conserved among human and mouse RRM2 proteinfamilies, is in close proximity to the RRM1/RRM2 interface. Thestructure of the ligand binding pocket is set forth in FIG. 4. Thepocket consists of helices α7, α8, and α10 at the C-terminal domain. Thenarrow interior end of the V-shaped pocket is lined up with hydrophobicresidues near the back of dityrosyl diiron cluster center. Polarresidues such as D271, R330, and E334 that are located near the open-endof the pocket may potentially interact with the flexible C-terminus. Thepocket is lined mostly with interior hydrophobic residues with chargedresidues exposed to the surface.

Compounds that docked into the ligand binding pocket were identifiedusing the TRIPOS FlexX docking tool and ranked using an embeddedconsensus docking score. The top 80 RR inhibitor candidates thatexhibited a binding affinity equal to or greater than that of 3-AP inthe virtual screen were subjected to an in vitro screen using a knownsemi-high throughput holoenzyme-based assay for determining the potencyand subunit-selectivity of small molecule inhibitors (Shao 2005). Theassay utilized recombinant RRM1/RRM2 or RRM1/RRM2 complex and measured[³H]CDP reduction activity (i.e., CDP to dCDP) by HPLC. Ten compoundsexhibited the ability to inhibit native RRM1/RRM2 activity by greaterthan 50% in vitro, and four of these compounds inhibited enzyme activityby 80% or more. Three of the four compounds exhibiting ≥80% inhibitionshared a similar structural scaffold (NCI-3, NSC #659390, and NSC#45382), and also showed better solubility and lower toxicity than theother tested compounds. NCI-3 (dihydroxyphenylthiazole, DHPT) has thefollowing structure:

A series of NCI-3 analogs were synthesized using the strategy set forthin FIG. 5. An additional 24 NCI-3 analogs were developed by attaching avariety of R groups to the aminothiazole group. Compounds generated inthis manner included COH1, COH2, COH4, COH20 and COH29.

Example 3: Characterization of Novel NCI-3 Analogs

The ability of NCI-3 and the NCI-3 analogs synthesized in Example 2 toinhibit RR activity was tested using the in vitro holoenzyme assaydescribed above. COH4 exhibited significant RR inhibition. COH20 waseven more effective, causing 90.2% inhibition of the recombinantRRM1/RRM2 complex in vitro (FIG. 6). IC50 results for various compoundsare set forth in Table 1.

TABLE 1 Compound IC50 ± S.D. (μM) HU 148.0 ± 7.34  3-AP  1.2 ± 0.13NCI-3 19.1 ± 0.43 COH4 15.3 ± 1.8  COH20 9.3 ± 2.3

In striking contrast to 3-AP, inhibition of RR by COH20 was virtuallyunaffected by the addition of iron (Table 2).

TABLE 2 IC50 ± S.D. (μM) RRM1/RRM2 COH20 alone 9.31 ± 2.3 COH20-Fecomplex 9.12 ± 1.9 COH20-Fe complex with added Fe 10.41 ± 2.1 

Site-directed mutagenesis, Biacore analysis, and NMR Saturation TransferDifference (STD) analysis were carried out to validate the bindingpocket and ligand/protein interaction between COH20 and RRM2. RRM2 pointmutants were generated by mutating certain key residues in the bindingpocket. These residues included Y323, D271, R330, and E334, each ofwhich are charged and reside on the surface of the binding pocket, andG233, which sits deep in the pocket. Attenuation of inhibition in thesemutants confirmed involvement of the mutated residues in ligand bindingand validated the binding pocket. Interestingly, the only mutation thatdid not attenuate inhibition was G233V. This suggests the presence of ahydrophobic pocket that is stabilized by introduction of a valine sidechain.

To confirm that the ability of COH20 to inhibit RR activity was notspecific to recombinant RR, an assay was performed testing the effect ofCOH20 on intracellular RR. KB cells treated with 10 μM COH20 were lysed,and protein was extracted in a high salt buffer and passed through a G25SEPHADEX® column to remove small molecules such as dNTP. The eluate wasmixed with [³H]CDP in reaction buffer to monitor RR activity. Treatmentwith COH20 decreased intracellular RR activity by approximately 50%(FIG. 7). Treatment with COH20 had no effect on RRM2 protein levels asmeasured by Western blot, indicating that the effect of COH20 on RRactivity is not due to a decrease in RRM2 expression.

dNTP pools from KB cells were measured by polymerase template assayfollowing treatment with 10 μM COH20. Pre- and post-treatment cellpellets were mixed with 100 μl of 15% trichloroacetic acid, incubated onice for ten minutes, and centrifuged at high speed for five minutes.Supernatants were collected and extracted with two 50 μl aliquots ofFreon/trioctylamine (55%/45%) to neutralize the trichloroacetic acid.After each addition, the samples were centrifuged at high speed andsupernatant was collected. Two 5 μl aliquots (one for each duplicate) ofeach sample were used to check dATP, dCTP, dGTP, and dTTPconcentrations. The reaction mixture in each tube contained 50 mMTris-HCL pH 7.5, 10 mM MgCl, 5 mM DTT, 0.25 mM template/primer, 1.25 μM³H-dATP (for dCTP assay) or ³H-dTTP (for dATP assay), and 0.3 units ofSEQUENASE™ (2.0) in a total volume of 50 μL. DNA synthesis was allowedto proceed for 20 minutes at room temperature. After incubation, 40 μlof each reaction mixture was spotted onto a WHATMAN® DE81 ion exchangepaper (2.4 cm diameter). The papers were dried for 30-60 minutes at roomtemperature, washed with 5% Na₂HPO₄ (3×10 minutes), and rinsed once withdistilled water and once more with 95% ethanol. Each paper was dried anddeposited in a small vial, and 5 ml of scintillation fluid was added toeach vial. Tritium-labeled dNTPs were counted using liquid scintillationcounter and compared to standards prepared at 0.25, 0.5, 0.75, and 1.0pmol/μL of dNTPs. For comparison, duplicate sets of reactions werecarried out with freshly added inhibitors. COH20 was found to decreasedATP, dCTP, dGTP, and dTTP pools in KB cells, indicating that inhibitionof RR results in a concomitant decreased in dNTP production (FIG. 8).Similar experiments will be performed using other cell lines.

The in vitro cytotoxicity of COH4 and COH20 towards human leukemia REHand MOLT-4 cells, human prostate cancer LNCaP cells, human oropharyngealcancer KB cells, and normal fibroblast NHDF cells was evaluated using anMTT assay. 5,000 cells were seeded on six-well plates for 72 hours withvarious concentrations of drug. COH20 was cytotoxic to the cancer celllines at less than 10 μM, while causing less cytotoxicity to normalcells than 3-AP. The results are summarized in Table 3. Results forLNCaP, KB, and NHDF are set forth in FIGS. 9-11. Based on the broadrange of cancer cell types against which COH20 exhibits cytotoxicity,COH20 is expected to be cytotoxic to a variety of additional cancer celltypes, including colon cancer, breast cancer, lung cancer, melanoma,leukemia, and lymphoma cells.

TABLE 3 IC50 (μM) Cell line COH20 COH4 3-AP HU REH 2.54 20.6 1.42 32.8MOLT-4 5.26 11.85 1.21 165 LNCaP 8.49 22.96 1.75 280 KB 9.25 30.6 1.98300 NHDF 82.8 52.6 7.35 >1000

In vitro cytotoxicity assays were repeated using KBHURs, an HU-resistantclone derived from KB cells that overexpresses RRM2. COH20 was cytotoxicto KBHURs at significantly lower concentrations than the other RRinhibitors tested, confirming that COH20 is capable of overcoming HUresistance (FIG. 12). In addition, COH20 was found to be cytotoxic toKBMDR, a KB clone that overexpresses the MDR pump on the cell membrane,at lower concentrations than 3-AP or HU (FIG. 13). A real-timeproliferation assay confirmed that COH20 also inhibits cellproliferation in KBMDR cells (FIG. 14). Similar experiments will berepeated using the gemcitabine resistant cell line KBGem, which alsooverexpresses RRM2. Based on the results with other cell lines, it isexpected that COH20 will also exhibit cytotoxicity and growth inhibitiontowards KBGem.

The in vitro cytotoxicity of COH29 towards a panel of human cancer celllines was tested using the MTT assay described above. COH29significantly inhibited growth across a broad range of cancer celltypes, with an IC50 of less than about 10 μM in all cell types testedexcept for colon cancer HT29, melanoma UACC-257, ovarian cancerNCI/ADR-RES, and renal cancer CAKI-1 (FIGS. 20-24). Representativeresults are summarized in Table 4.

TABLE 4 Cell line IC50 Leukemia CCRF-CEM 2.8 μM Leukemia MOLT-4 2.5 μMLeukemia SUP B15 5.0 μM Ovarian Cancer OV 90 2.6 μM

Flow cytometry and annexin staining were performed on KB cells treatedwith COH20 at 9 or 27 μM or 3-AP at 3 μM for 24 hours. These resultsshowed that COH20 treatment arrests cells in S-phase in a dose-dependentmanner (FIG. 15A-15D). After treatment with COH20 for 72 hours, annexinstaining showed significant cell death, indicating apoptosis (FIGS.15E-15H). COH20 induced apoptosis with approximately the same potency as3-AP.

COH20 was injected into three male rats at 1 mg/kg for single-dosepharmacokinetic evaluation. Elimination of COH20 from plasma was foundto be tri-exponential, with a rapid initial decline phase (possibletissue distribution or liver uptake) followed by an intermediate phase(combined distribution and elimination) and a slower terminal phase(elimination) (FIG. 16). The terminal half-life (T_(1/2)) wasapproximately 5.5 hours. More detailed pharmacokinetic studies will beperformed with various dosages of COH20 to establish parameters such asclearance, bioavailability, and tissue/plasma partition coefficients.

Pharmacokinetic evaluation was performed on COH29 using the sametechniques, with COH29 administered at a dosage of 25 mg/kg. Resultsfrom triplicate analysis are summarized in Table 5. Area under the curve(AUC) calculations showed COH29 acting in a dose-dependent manner whenadministered by i.v. bolus (FIG. 25).

TABLE 5 C_(max) T_(max) AUC CL V_(ss) T_(1/2) (ng/ml) (hr) (ng*h/ml)(ml/(h*kg)) (ml/kg) (hr) 1 31300 0.03 3668 6816 1185 0.4 2 26000 0.032861 8738 576 7.6 3 25600 0.03 3918 6381 653 0.03 Avg. 27633 0.03 34827312 804 2.67 SD 1837 — 319 724 191 2.45

In order to determine the maximal tolerated dose of COH20, COH20 wasadministered to mice intravenously at dosages ranging from 10 to 160mg/kg. Other than one mouse that died in the 160 mg/kg group, bodyweight remained stable for all treatment groups, indicating that COH20is a tolerable compound with minimal toxicity (FIG. 17). There was noevidence of lethal iron chelation or induction of methemoglobulin,further indicating that COH20 has no significant iron chelation sideeffects.

A Biacore T100 instrument was used to study the ligand-proteininteraction between COH20 or 3-AP and RRM2. Wild-type RRM2 was isolatedand immobilized onto CM4 sensor chips using standard amine-couplingmethods at 25° C. and a flow rate of 10 μL/minute. Specifically, thecarboxymethyl dextran surfaces of the flow cells were activated with a7-min injection of a 1:1 ratio of 0.4 M(N-ethyl-NO-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.1 MN-hydroxysuccinimide (NHS). RRM2 was diluted in 10 mM sodium acetate, pH4.5, to 25 μg/ml and injected over target flow cell for targetimmobilization 8000RU. 10 mM sodium acetate, pH 4.5 buffer was injectedinto the reference flow cell for blank immobilization. The remainingactivated surface was blocked with a 7-min injection of 1Methanolamine-HCl, pH 8.5. Phosphate-buffered saline (PBS) was used as arunning buffer during immobilization. COH20 and 3-AP were dissolved inDMSO to prepare 100 mM stock solutions. The compounds were then dilutedserially in running buffer (PBS, 1.5% DMSO, 10-fold carboxymethyldextran, 0.002% Methyl-6-O—(N-heptylcarbamoyl)-α-D-glucoyranoside) tothe appropriate running concentrations. Samples were injected over thereference flow cell and target flow cell (with immobilized RRM2) at aflow rate of 60 μL/min at 25° C. Association and dissociation weremeasured for 180 seconds and 60 seconds, respectively. All compoundswere tested in triplicate at five different concentrations.Concentration series for each compound were done at least twice. Withina given compound concentration series, the samples were randomized tominimize systematic errors. Between samples, the sensor chip wasregenerated by injection of 0.3% SDS for 30 seconds. The results showeda significant interaction between COH20 and RRM2, but not between COH20and 3-AP (FIG. 18).

The Biacore T100 was also used to analyze the ability of COH20 tointerfere with binding of RRM2 to RRM1. Fixed concentrations of RRM1 (1μM) in the absence and presence of a two-fold dilution concentrationseries of COH20 (3.125-25 μM) were injected over a reference flow celland a target flow cell (with immobilized RRM2) at a flow rate 30 μL/minat 25° C. Association and dissociation were measured for 90 seconds and60 seconds, respectively. Duplicates runs were performed using the sameconditions. Between samples, the sensor chip was regenerated byinjection of 0.3% SDS, 0.2 M Na₂CO₃ for 30 seconds. COH20 was found tointerrupt the RRM1/RRM2 interaction at the interface (FIG. 19).

The cytotoxic efficacy of COH20 will be tested in vivo using a mousexenograft model. Xenograft tumor models will be created using humancancer cell lines such as KB, KBHURs, and KBGem. For establishment ofthe KB xenograft model, 1-5 10⁶ KB cells in a volume of 0.1 ml salinewill be injected into the right hind flank of 5-6 week old nude femalemice. Tumor volume will be monitored twice weekly using digitalcalipers. When tumor volume reaches approximately 100-160 mm³, mice willbe divided into groups of ten such that the median and mean body weightand tumor volume are roughly the same for all mice within a group. COH20will be administered either 1) alone in a single dose to determineeffective dosage, 2) alone at various intervals for a scheduling study,or 3) in combination with known cancer therapeutics such aschemotherapeutics. During a monitoring period of approximately fourweeks, changes in tumor cell growth, body weight, organ dysfunction, andiron chelating side effects will be analyzed at various timepoints.Following the monitoring period, mice will be euthanized and tissue,tumor, and plasma will be analyzed by visual and histologicalexamination. Based on the cytotoxicity of COH20 towards various cancercell lines in vitro, it is expected that mice treated with COH20 willexhibit higher survival rates, decreased tumor growth, and fewertumor-related side effects (e.g., weight loss, organ dysfunction).

The structure of NCI-3 analogs such as COH20 and COH29 may be refinedand optimized by generating various analogs and analyzing their bindingto RRM2 and the RRM1/RRM2 complex using site-directed mutagenesisstudies, Biacore analysis, and NMR STD experiments. X-raycrystallography studies may be performed to determine thethree-dimensional structure of the COH20-RRM2 complex. Using thesetools, additional RR inhibitors may be generated with higher potency,greater selectivity, and lower toxicity.

As shown above, the RRM2 mutant G233V enhanced COH20 inhibitor activity.Additional hydrophobic interactions between the valine side chain andthe bound ligand are believed to contribute to enhanced binding andinhibition, suggesting that an additional hydrophobic side chainextending from COH20 could optimize binding affinity. Therefore, COH20analogs that contain these hydrophobic side chains (e.g. COH1, COH2,COH4, COH29, compounds having Structure I, and compounds selected fromGroup I) are also likely to be RR inhibitors.

Example 4: Synthesis and Purification of COH29

COH29 was synthesized using the synthesis pathway outlined in FIG. 26.

Step 1: Conversion of 1,2-dimethoxybenzene (veratrole) to1-(3,4-dimethoxyphenyl)-2-phenylethanone (Intermediate 1)

Intermediate 1 was synthesized in a reaction vessel equipped with abubbler vented to a cold finger trap. Phenylacetyl chloride (151 mL,1.12 mol) was added drop-wise over 30 minutes to a stirring suspensionof anhydrous AlCl₃ (161 g, 1.21 mol) in dichloromethane (DCM; CH₂Cl₂,600 mL) at 0° C. under nitrogen. Veratrole (129 mL, 1.00 mol) was addeddrop-wise over six hours, maintaining the internal temperature below 10°C. Upon completion of addition, the cooling bath was removed. After 16hours at ambient temperature, the reaction was cooled to 0° C. andquenched by drop-wise addition of 2N HCl (700 mL) while maintaining theinternal temperature below 20° C. The organic layer was washed withwater (600 mL) and saturated aqueous NaHCO₃ (500 mL), filtered throughCELITE®, and concentrated under reduced pressure to approximately 400 mLtotal volume. Hexanes (1.7 L) were added and the product precipitatedwith vigorous stirring. The resulting solid was filtered and the filtercake was washed with hexanes (2×250 mL). The solid was dried in vacuo at50° C. to afford 1-(3,4-dimethoxyphenyl)-2-phenylethanone (Intermediate1; 213 g, 831 mmol, 83% yield) as an off-white solid.

Step 2: Conversion of Intermediate 1 to4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-amine (Intermediate 2)

Intermediate 2 was synthesized in a reaction vessel equipped with abubbler vented to a cold finger trap. Intermediate 1 (213 g, 831 mmol)and pyridinium tribromide (315 g, 886 mmol) were dissolved in DCM (1.2L) under nitrogen. After five hours at ambient temperature, the reactionmixture was cooled in an ice batch and quenched with water (750 mL)while maintaining the internal temperature below 20° C. The organiclayer was washed with water (750 mL) and concentrated to dryness. Theresulting slurry was taken up in ethanol (1.2 L) and cooled to 20° C.,and thiourea (114 g, 1.48 mol) was added. Upon addition completion, thereaction was stirred at ambient temperature for 24 hours. The reactionwas concentrated under reduced pressure and the resulting slurry waspartitioned between EtOAc (1.0 L) and 2N NaOH (800 mL). The emulsion wasallowed to separate, and the aqueous layer was extracted with EtOAc (750mL). The combined organic layers were washed with water (250 mL) andconcentrated under reduced pressure. The resulting solid was trituratedwith Et₂O (1.5 L) and filtered, and the filter cake was washed with Et₂O(200 mL). The product was dried in vacuo at 50° C. to afford4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-amine (Intermediate 2; 230 g,736 mmol, 89% yield for 2 steps) as a tan solid.

Step 3: Conversion of Intermediate 2 toN-(4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-yl)-3,4-dimethoxybenzamide(Intermediate 3)

Intermediate 3 was synthesized in a reaction vessel equipped with abubbler vented to a cold finger trap. Thionyl chloride (100 mL, 1.36mol) was added drop-wise over two hours to a cooled solution of3,4-dimethoxybenzoic acid (250 g, 1.36 mol) in dimethylformamide (DMF;20 mL) in CH₂Cl₂ (1.0 L) while maintaining an internal temperature below15° C. After addition completion, the reaction was stirred for threehours at ambient temperature, then concentrated to dryness under reducedpressure. Acid chloride (164 g, 818 mmol, 1.5 eq) was added portion-wiseto a stirring suspension of Intermediate 2 (170 g, 545 mmol),4-dimethylamino pyridine (4-DMAP; 6.73 g, 54.5 mmol, 10 mol %), and Et₃N(384 mL, 2.73 mol) in DMF (1.0 L) under nitrogen, maintaining aninternal temperature below 20° C. The reaction was stirred for 15 hoursat ambient temperature, then the remaining acid chloride (112 g, 545mmol) was portion-wise. After six hours at ambient temperature, thereaction mixture was quenched with saturated aqueous sodium NaHCO₃ (500mL), washed with water (500 mL), and concentrated under reducedpressure. The product was purified by flash chromatography (0-5%MeOH/CH₂Cl₂. The combined chromatographic fractions were concentrated todryness under reduced pressure. The solid was dried in vacuo at 50° C.to affordN-(4-(3,4-dimethoxyphenyl)-5-phenylthiazol-2-yl)-3,4-dimethoxybenzamide(Intermediate 3; 245 g, 515 mmol, 70%) as an off-white solid.

Step 4: Conversion of Intermediate 3 to COH29

COH29 was synthesized in a reaction vessel equipped with a bubblervented to a cold finger trap. Boron tribromide (BBr₃; 191 mL, 1.98 mol)was slowly added to a stirring solution of Intermediate 3 (245 g, 515mmol) in toluene (1.2 L) under nitrogen, maintaining the internaltemperature below 15° C. Upon addition completion, the reaction wasallowed to warm to ambient temperature and stirred for five hours. Thereaction mixture was cooled and slowly quenched with EtOH (800 mL) whilemaintaining the internal temperature below 20° C. Upon additioncompletion, the solution was stirred for two hours at ambienttemperature and concentrated under reduced pressure. The resulting solidwas triturated with DCM (1.0 L) and filtered, and the filter cake waswashed with DCM (100 mL). The resulting solid was taken up in hot EtOH(400 mL) and slowly added to water (2.4 L) to induce precipitation. Theresulting slurry was stirred for two hours at ambient temperature andfiltered, and the filter cake was washed with water (200 mL). Thistrituration process was repeated three additional times to afford anoff-white solid. The resulting solid was dried at 50° C. under vacuumfor 24 hours, then at 125° C. under vacuum for 24 hours to afford COH29(118 g, 281 mmol, 55%)

As stated above, the foregoing are merely intended to illustrate thevarious embodiments of the present invention. As such, the specificmodifications discussed above are not to be construed as limitations onthe scope of the invention. It will be apparent to one skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is understoodthat such equivalent embodiments are to be included herein. Allreferences cited herein are incorporated by reference as if fully setforth herein.

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1.-15. (canceled)
 16. A compound having the formula

wherein, R is substituted or unsubstituted aryl, R₁, R₂, R₃, and R₇ areindependently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen,R₁₇-substituted or unsubstituted alkyl, R₁₇-substituted or unsubstitutedheteroalkyl, R₁₇-substituted or unsubstituted cycloalkyl,R₁₇-substituted or unsubstituted heterocycloalkyl, R₁₇-substituted orunsubstituted aryl, or R₁₇-substituted or unsubstituted heteroaryl, R₁₇is —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₂₀-substituted orunsubstituted alkyl, R₂₀-substituted or unsubstituted heteroalkyl,R₂₀-substituted or unsubstituted cycloalkyl, R₂₀-substituted orunsubstituted heterocycloalkyl, R₂₀-substituted or unsubstituted aryl,or R₂₀-substituted or unsubstituted heteroaryl, R₂₀ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, R₂₁-substituted or unsubstituted alkyl,R₂₁-substituted or unsubstituted heteroalkyl, R₂₁-substituted orunsubstituted cycloalkyl, R₂₁-substituted or unsubstitutedheterocycloalkyl, R₂₁-substituted or unsubstituted aryl, orR₂₁-substituted or unsubstituted heteroaryl, and R₂₁ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.
 17. The compound ofclaim 16, wherein R is substituted aryl.
 18. The compound of claim 16,wherein R₁, R₂, R₃, and R₇ are independently hydrogen, —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, or halogen.
 19. The compound of claim 16, whereinR₁, R₂, R₃, and R₇ are independently hydrogen.
 20. The compound of claim16, wherein R₁, R₂, R₃, and R₇ are independently R₁₇-substituted orunsubstituted alkyl, R₁₇-substituted or unsubstituted heteroalkyl,R₁₇-substituted or unsubstituted cycloalkyl, R₁₇-substituted orunsubstituted heterocycloalkyl, R₁₇-substituted or unsubstituted aryl,or R₁₇-substituted or unsubstituted heteroaryl.
 21. The compound ofclaim 16, wherein R₁, R₂, R₃, and R₇ are independently R₁₇-substitutedor unsubstituted alkyl, R₁₇-substituted or unsubstituted heteroalkyl.22. A method of synthesizing a compound having the formula:

wherein said method comprising contacting a compound having the formula

with a compound having the formula

wherein, R is substituted or unsubstituted aryl, R₁, R₂, R₃, R₄, R₅, R₆and R₇ are independently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo,halogen, R₁₇-substituted or unsubstituted alkyl, R₁₇-substituted orunsubstituted heteroalkyl, R₁₇-substituted or unsubstituted cycloalkyl,R₁₇-substituted or unsubstituted heterocycloalkyl, R₁₇-substituted orunsubstituted aryl, or R₁₇-substituted or unsubstituted heteroaryl, R₈,R₉, R₁₀ and R₁₁ are independently a hydroxyl protecting group, whereinR₈ and R₉ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl and R₁₀ and R₁₁ are optionally joinedtogether to form a substituted or unsubstituted heterocycloalkyl, R₁₇ is—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₂₀-substituted orunsubstituted alkyl, R₂₀-substituted or unsubstituted heteroalkyl,R₂₀-substituted or unsubstituted cycloalkyl, R₂₀-substituted orunsubstituted heterocycloalkyl, R₂₀-substituted or unsubstituted aryl,or R₂₀-substituted or unsubstituted heteroaryl, R₂₀ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, R₂₁-substituted or unsubstituted alkyl,R₂₁-substituted or unsubstituted heteroalkyl, R₂₁-substituted orunsubstituted cycloalkyl, R₂₁-substituted or unsubstitutedheterocycloalkyl, R₂₁-substituted or unsubstituted aryl, orR₂₁-substituted or unsubstituted heteroaryl, and R₂₁ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.
 23. The method of claim22, wherein R is substituted aryl.
 24. The method of claim 22, whereinR₁, R₂, R₃, R₄, R₅, R₆ and R₇ are independently hydrogen, —OH, —NH₂,—SH, —CN, —CF₃, —NO₂, oxo, or halogen.
 25. The method of claim 22,wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are hydrogen.
 26. The method ofclaim 22, wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are independentlyR₁₇-substituted or unsubstituted alkyl, R₁₇-substituted or unsubstitutedheteroalkyl, R₁₇-substituted or unsubstituted cycloalkyl,R₁₇-substituted or unsubstituted heterocycloalkyl, R₁₇-substituted orunsubstituted aryl, or R₁₇-substituted or unsubstituted heteroaryl. 27.The method of claim 22, wherein R₈, R₉, R₁₀ and R₁₁ are independentlysubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, or a substituted silyl.28. The method of claim 22, wherein R₈, R₉, R₁₀ and R₁₁ areindependently an activated ethylene protecting group, a benzyl etherprotecting group, or a silicon-based carbonate protecting group.
 29. Themethod of claim 22, wherein R₈ and R₉ are optionally joined together toform an acetal protecting group and R₁₀ and R₁₁ are optionally joinedtogether to form an acetal protecting group.
 30. The method of claim 22,wherein said acetal is a diphenyl methylene acetal.
 31. A method ofusing a first compound having the formula:

wherein, R is substituted or unsubstituted aryl, R₁, R₂, R₃, R₄, R₅, R₆and R₇ are independently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo,halogen, R₁₇-substituted or unsubstituted alkyl, R₁₇-substituted orunsubstituted heteroalkyl, R₁₇-substituted or unsubstituted cycloalkyl,R₁₇-substituted or unsubstituted heterocycloalkyl, R₁₇-substituted orunsubstituted aryl, or R₁₇-substituted or unsubstituted heteroaryl, R₈,R₉, R₁₀ and R₁₁ are independently a hydroxyl protecting group, whereinR₈ and R₉ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl and R₁₀ and R₁₁ are optionally joinedtogether to form a substituted or unsubstituted heterocycloalkyl, R₁₇ is—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₂₀-substituted orunsubstituted alkyl, R₂₀-substituted or unsubstituted heteroalkyl,R₂₀-substituted or unsubstituted cycloalkyl, R₂₀-substituted orunsubstituted heterocycloalkyl, R₂₀-substituted or unsubstituted aryl,or R₂₀-substituted or unsubstituted heteroaryl, R₂₀ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, R₂₁-substituted or unsubstituted alkyl,R₂₁-substituted or unsubstituted heteroalkyl, R₂₁-substituted orunsubstituted cycloalkyl, R₂₁-substituted or unsubstitutedheterocycloalkyl, R₂₁-substituted or unsubstituted aryl, orR₂₁-substituted or unsubstituted heteroaryl, and R₂₁ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl, in the preparation ofone or more additional compounds in one or more additional steps. 32.The method of claim 31, further comprising the formation of apharmaceutical product.
 33. The method of claim 31, wherein one or moreadditional compounds is a compound having the formula:


34. A method of using a first compound having the formula:

wherein, R is substituted or unsubstituted aryl, R₁, R₂, R₃, and R₇ areindependently hydrogen, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen,R₁₇-substituted or unsubstituted alkyl, R₁₇-substituted or unsubstitutedheteroalkyl, R₁₇-substituted or unsubstituted cycloalkyl,R₁₇-substituted or unsubstituted heterocycloalkyl, R₁₇-substituted orunsubstituted aryl, or R₁₇-substituted or unsubstituted heteroaryl, R₁₇is —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, R₂₀-substituted orunsubstituted alkyl, R₂₀-substituted or unsubstituted heteroalkyl,R₂₀-substituted or unsubstituted cycloalkyl, R₂₀-substituted orunsubstituted heterocycloalkyl, R₂₀-substituted or unsubstituted aryl,or R₂₀-substituted or unsubstituted heteroaryl, R₂₀ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, R₂₁-substituted or unsubstituted alkyl,R₂₁-substituted or unsubstituted heteroalkyl, R₂₁-substituted orunsubstituted cycloalkyl, R₂₁-substituted or unsubstitutedheterocycloalkyl, R₂₁-substituted or unsubstituted aryl, orR₂₁-substituted or unsubstituted heteroaryl, and R₂₁ is —OH, —NH₂, —SH,—CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl, in preparation of asecond compound.
 35. The method of claim 34, wherein said secondcompound is a pharmaceutical product.
 36. The method of claim 34,wherein said second compound is a compound having the formula:

wherein R₄, R₅, and R₆ are independently hydrogen, —OH, —NH₂, —SH, —CN,—CF₃, —NO₂, oxo, halogen, R₁₇-substituted or unsubstituted alkyl,R₁₇-substituted or unsubstituted heteroalkyl, R₁₇-substituted orunsubstituted cycloalkyl, R₁₇-substituted or unsubstitutedheterocycloalkyl, R₁₇-substituted or unsubstituted aryl, orR₁₇-substituted or unsubstituted heteroaryl, and R₈, R₉, R₁₀ and R₁₁ areindependently a hydroxyl protecting group, wherein R₈ and R₉ areoptionally joined together to form a substituted or unsubstitutedheterocycloalkyl and R₁₀ and R₁₁ are optionally joined together to forma substituted or unsubstituted heterocycloalkyl.